Antibodies have long been a mainstay of biological and medical research, and current use of antibodies as therapeutics has further expanded their portfolio of applications. More recently, to address various challenges such as the reduced stability and production yields of the antibody fragments that are frequently employed in in vitro evolution platforms, and in large part as a result of intellectual property concerns, the field of alternative binding scaffolds has emerged (Skerra A., Curr. Opin. Biotechnol., 18:295-304, 2007). By mutagenizing solvent-exposed loop regions or inserting diverse loop repertoires into non-antibody protein scaffolds, specific binding attributes can be conferred to proteins that naturally have desirable properties such as high stability and production titers. In this way, alternative scaffolds such as the 10th human fibronectin type III domain (Lipovsek D et al., Journal of Molecular Biology, 368:1024-1041, 2007), anticalins (Korndorfer I P et al., Journal of Molecular Biology, 330:385-396, 2003; Schlehuber S et al., Journal of Molecular Biology, 297:1105-1120, 2000; Vogt M & Skerra A, Chembiochem 5:191-199, 2004), designed ankyrin repeat proteins (Zahnd C et al., Journal of Molecular Biology, 369:1015-1028, 2007), and Affibodies (Nord K et al., Eur J Biochem, 268:4269-4277, 2001; Nord K et al., Nat Biotechnol, 15:772-777, 1997), among others, have been developed to bind to targets with antibody-like affinity.
Green fluorescent protein (GFP) has also been explored as a potential alternative scaffold. To date, GFP has been utilized for a wide variety of different applications (Zhang J et al., Nat Rev Mol Cell Biol, 3:906-918, 2002) including Ca2+ detection (Miyawaki A et al., Nature, 388:882-887, 1997), visualization of protein-protein interactions (Hu C D & Kerppola T K, Nature Biotechnology, 21:539-545, 2003), and as a reporter for protein folding (Waldo G S et al., Nature Biotechnology, 17:691-695, 1999). Considerable effort has also been expended in attempts to develop GFP as a binding scaffold that would have two potential advantages over the aforementioned alternative scaffolds. First, by combining binding attributes with the intrinsic fluorescence of the GFP protein, the proteins could act as single step detection reagents in applications such as fluorescence-based ELISAs, flow cytometry, and intracellular targeting/trafficking in live cells. Second, GFP fluorescence requires that the protein is properly folded (Reid B G & Flynn G C, Biochemistry, 36:6786-6791, 1997) offering an in situ metric for folding fidelity, absent from other alternative scaffolds. Such a folding probe could assist both assessment of library fitness upon binding loop introduction, and subsequent selection of properly-folded, soluble clones.
Several attempts have been made to confer binding capability to GFP by inserting binding loops into various solvent-exposed turns that connect the β-strands of the GFP β-barrel structure. The regions of GFP that are most amenable to insertion of amino acids have been determined (turns Gln157-Lys158 and Glu172-Asp173) (Abedi M R et al., Nucleic Acids Research, 26:623-630, 1998; Doi N & Yanagawa H, Febs Letters, 453:305-307, 1999), although fluorescence is diminished substantially, and when random loops were inserted, the resultant library fluorescence decreased to 2.5% of wild-type (Abedi M R et al., Nucleic Acids Research, 26:623-630, 1998). Selection of GFP-inserted peptide libraries for targeting various intracellular compartments has also been performed (Peelle B et al., Chem Biol, 8:521-534, 2001).
GFP-inserted peptide libraries have also been selected to identify peptides useful for targeting various intracellular compartment. Antibody heavy chain CDR3 sequences have been inserted into several loop regions of superfolder GFP, a GFP variant evolved for high stability and improved folding kinetics (Pedelacq J D et al., Nature Biotechnology, 24:79-88, 2006), to create libraries of single CDR3-inserted GFP. Results from this study indicated that insertion at many sites substantially reduces GFP fluorescence as seen previously with standard GFP variants (Kiss C et al., Nucleic Acids Research, 34:15, 2006). Three loop regions of the superfolder GFP, however, tolerated single loop CDR insertions (including Asp173-Gly174) such that it was possible to isolate fluorescent binders against protein targets using T7 phage display, with the best being a 470 nM lysozyme binder (Dai M et al., Protein Engineering Design & Selection, 21:413-424, 2008). This level of affinity is in the realm of that found for peptide binders (Craig L et al., J Mol Biol, 281:183-201, 1998) likely as a consequence of its single binding loop design. Affinity of GFP-based binding proteins could therefore in principle benefit from display of multiple binding loops which could act together to form a cooperative binding interface. However, the lone examples of multiple loop insertion into GFP include insertion of haemagglutinin peptide (Zhong J Q et al., Biomolecular Engineering, 21:67-72, 2004) or random loops (Chen S-S et al., Biochem. Biophys. Res. Commun., p. doi:10.1016/j.bbrc.2008, 2008).1006.1123 into two loops on opposite faces of GFP. While suitable for the authors' goals, these insertion locations would not be ideal for forming a cooperative binding interface. Moreover, GFP fluorescence of the resulting clones in the case of the random loop libraries was not demonstrated (Chen S-S et al., Biochem. Biophys. Res. Commun., p. doi:10.1016/j.bbrc.2008, 2008).
Thus, to date, robust fluorescent multiple loop-inserted GFP repertoires have not been described, even using the superfolder GFP as a template.